Genetically purified gellan gum

ABSTRACT

Mutational inactivation of proteins involved in para-cresol production in certain milk products results in improved taste and odor. The undesirable para-cresol forms over time as a result of enzymes produced by the bacterium that produces gellan gum. Since the gellan is typically used in a relatively unpurified form, the enzymes are added to the milk along with the gellan. Inactivation of the enzymes is a genetic means of eliminating the enzymes without requiring any additional purification or processing.

This application claims priority under 35 U.S.C. 119(e) to provisionalapplication 60/580,730 filed on Jun. 21, 2004.

A portion of the disclosure of this patent document contains materialwhich is subject to copyright protection. The copyright owner has noobjection to the facsimile reproduction by anyone of the patent documentor the patent disclosure, as it appears in the Patent and TrademarkOffice patent file or records, but otherwise reserves all copyrightrights whatsoever.

FIELD OF THE INVENTION

The invention relates to the field of food additives. In particular itrelates to the field of dairy food additives. More particularly itrelates to additives to sterilized milk products.

BACKGROUND OF THE INVENTION

Gellan gum is an extracellular polysaccharide produced by the bacteriaSphingomonas elodea. Gellan gum produced by S. elodea is commerciallyavailable as Kelcogel LT100® from CP Kelco, San Diego, Calif.Commercially, gellan gum is formed by aerobic fermentation. Uponcompletion of fermentation, the broth is pasteurized to kill viablecells prior to recovery of the gum from the fermentation broth.

Gellan gum comprises the sugars glucose, glucuronic acid, and rhamnosein a 2:1:1 molar ratio, which are linked to form a tetrasacchariderepeat unit. Native gellan gum is acetylated and glycerylated on thesame glucose residues. On average, there is one acetyl group and onehalf glyceryl group per tetrasaccharide repeat unit.

The method of recovery of the gellan gum affects the characteristics ofthe gum. Direct recovery yields a soft, flexible gel. Gellan gum haslong been used in cultured, retorted, and frozen dairy products due toits textural and rheological properties. However, an off-flavor and odordevelop in otherwise shelf-stable, milk-based, gellan-containingproducts; this flavor and odor render the foods unpalatable. Theoff-flavor and odor have been linked to the formation of para-cresolfrom substrates in milk, e.g., para-cresyl sulfate and para-cresylglucouronide. Para-cresol is detectable in milk-based, gellan-containingproducts that have been treated at ultra high temperatures and stored atroom temperature.

In an effort to eliminate this problem, gellan has been deacylated withhot alkali treatment. While effective in eliminating the para-cresol,the deacylation processing makes the gellan gum more brittle and lessuseful for certain food applications. Another approach to eliminate thisproblem is the pre-treatment of native gellan gum with a denaturingagent, such as sodium hypochlorite or potassium hydroxide. This approachadds material and processing costs. There is a need in the art for agellan product which does not produce para-cresol upon prolonged storagein a sterilized dairy product and which does not require extraprocessing steps.

BRIEF SUMMARY OF THE INVENTION

In a first embodiment a composition is provided which comprises gellangum substantially free of arylsulfatase protein.

In a second embodiment a composition is provided which comprises gellangum substantially free of β-glucuronidase protein.

In a third embodiment of the invention a composition is provided whichcomprises gellan gum substantially free of both arylsulfatase andβ-glucuronidase proteins.

In a fourth embodiment of the invention a method is provided forproducing a gellan gum composition. Sphingomonas elodea is cultured in aculture medium. The Sphingomonas elodea produces no catalytically activearylsulfatase, or no catalytically active β-glucuronidase, or nocatalytically active arylsulfatase and no catalytically activeβ-glucuronidase. The culture medium is collected. Gellan gum isprecipitated from the culture medium.

A microbiologically pure culture of Sphingomonas elodea is provided in afifth embodiment of the invention. It is arylsulfatase-deficient.

Another microbiologically pure culture of Sphingomonas elodea isprovided in a sixth embodiment of the invention. It isβ-glucuronidase-deficient.

Still another embodiment of the invention is a microbiologically pureculture of Sphingomonas elodea. It is deficient in both arylsulfataseand β-glucuronidase.

An eighth embodiment of the invention provides an isolated and purifiedpolynucleotide encoding a Sphingomonas elodea arylsulfatase. Thearylsulfatase has an amino acid sequence according to SEQ ID NO: 2.

A ninth embodiment of the invention provides an isolated and purifiedpolynucleotide encoding a Sphingomonas elodea β-glucuronidase. Theβ-glucuronidase has an amino acid sequence according to SEQ ID NO: 5.

A tenth embodiment of the invention is an isolated and purifiedpolynucleotide comprising Sphingomonas elodea genomic DNA. The genomicDNA comprises a deletion of all or part of its arylsulfatase codingsequence.

An eleventh embodiment of the invention is an isolated and purifiedpolynucleotide comprising Sphingomonas elodea genomic DNA. The genomicDNA comprises a deletion of all or part of its β-glucuronidase codingsequence.

These and other embodiments of the invention as described in more detailbelow provide the art with cost-effective means to make a moreconsumer-acceptable, sterilized, gellan-containing, dairy product.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Genetic map of the genomic region around the arylsulfatase gene(atsA) and location of the regions amplified by PCR and cloned intoplasmid pLO2. Plasmid pLO2 with the cloned PCR fragments was then usedto replace this region of the genome with the deletion, by homologousrecombination.

FIG. 2. Restriction map of the genomic region around thebeta-glucuronidase gene (gusA) of Sphingomonas elodea. Positions oftransposon insertions in clones BG-6 and BG-7 are indicated at thebottom.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have found that if either of the genes encodingthe enzymes arylsulfatase and β-glucuronidase or both genes aremutationally inactivated in the bacterium Sphingomonas elodea, thebacterium produces a gellan that has superior properties for certainpurposes. In particular, the gellan that is produced by such mutantsimparts to sterilized milk products a longer shelf-life.

If one or both of the enzymes are not inactivated then they producepara-cresol from substrates (p-cresyl-sulfate and p-cresyl-glucuronide)found in the milk. The para-cresol imparts an odor and flavor that isgenerally unpalatable to consumers. Eliminating these enzymes reducesthe rate at which para-cresol is produced in the sterilized milk or milkproduct on the shelf.

Mutations in either or both of the enzymes may be used to reduce therate of para-cresol production. The mutations are preferably of the typethat totally inactivates the protein, such as insertions, nonsense,frameshift, or deletion mutations. Any technique known in the art forproducing such mutations may be used. The applicants used a transposoninsertion strategy to identify the genes encoding arylsulfatase andβ-glucuronidase. A deletion mutation in each gene was then constructedby homologous recombination using 5′ and 3′ DNA fragments flanking thegene which were joined together. Nonetheless, other strategies can beused to obtain the mutations in these genes. The mutations can be madedirectly in a gellan “production” strain, or the mutations can betransferred to such a strain from a strain in which the mutation isfirst made. Techniques for site-directed mutagenesis are well known inthe art. See, e.g., “In Vitro Mutagenesis Protocols, second edition,Braman, Jeff, ed., Humana Press, 2002, and the commercially availableQuikChange™ kit (Stratagene). Provided with the wild-type sequences ofthe S. elodea arylsulfatase and β-glucuronidase genes, one of skill inthe art can readily make a variety of desired mutations in these genes.

The sequence of the wild-type and mutant genes encoding arylsulfataseand β-glucuronidase have been determined. See SEQ ID NO: 1 (wild-typearylsulfatase), SEQ ID NO: 3 (deletion of arylsulfatase), SEQ ID NO: 4(wild-type β-glucuronidase), and SEQ ID NO: 6 (deletion ofβ-glucuronidase). The identification of these genes and their nucleotidesequences permits one of skill in the art to readily make othermutations having the desired null phenotype for expression of theseenzymes. Mutations such as insertions, deletions, nonsense, andframeshift are most likely to result in a null phenotype for theenzymes. Missense mutations can also be made and routinely tested fortheir effect on enzyme activity. Standard techniques of microbialgenetics can be used to make suitable mutations. See, e.g., Principlesof Gene Manipulation: An Introduction to Genetic Engineering, R. W. Old,S. B. Primrose, Blackwell Publishing, 1994. Standard enzyme assays canbe used to test for loss of activity of the mutated enzymes. See, e.g.,Kim et al., Appl Microbiol Biotechnol. 2004 February; 63(5):553-9.

Compositions of the present invention which are substantially free ofarylsulfatase or β-glucuronidase contain less than 95%, 96%, 97%, 98%,or 99% of the amount of the particular protein than is contained inwild-type strains. Such a reduction in amount of enzyme should lead tosterilized milk compositions which have less than 90%, 93%, 95%, 97%, or99% of the amount of para-cresol that is produced in compositionscontaining gellan from wild-type strains. The amount of arylsulfatase orβ-glucuronidase protein which is produced can be measured by enzymeassay using a readily assayable substrate such as5-bromo-4-chloro-3-indolyl sulfate (X—SO4); CAS No. 6578-07-0 from Sigmaor using 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-GlcA); CAS No.114162-64-0, from Sigma or RPI Corp. or using p-nitrocatechol.Alternatively the protein can be measured using an immunologicaltechnique such as a Western blot.

Gellan gum is typically used in sterilized or ultra high temperature(UHT) treated dairy products or frozen dairy products. Such productsinclude without limitation, ice cream, frozen yogurt, pudding, whippeddairy product, coffee creamer, crème brulee, and dairy beverages. Thegellan gum of the present invention can be used in these or any otherfoods as can typical gellan from a wild-type strain. Gellan gum istypically used for suspension of fine particles, but it also can be usedto impart a favorable mouth feel.

Gellan gum can be produced using the mutant strains of the presentinvention according to any of the methods known in the art for wild-typestrains. The bacteria are typically grown in a liquid culture medium.Such medium typically contains a carbon source, phosphate, organic andinorganic nitrogen sources, and appropriate trace elements. Thefermentation is typically conducted under sterile conditions, withaeration and agitation. At the end of the fermentation period, theculture medium is collected, typically without removing the cells. Thefermentation broth can be pasteurized to kill viable cells prior torecovery of the gellan gum. The gellan gum can be precipitated as isknown in the art. Typically this is done with an alcohol, such asisopropanol. The precipitated gellan can be dried prior to rehydration.

Para-cresol can be measured by any means known in the art. One methodwhich can be used employs dichloromethane extraction, concentration, andgas chromatographic-mass spectroscopy.

While the invention has been described with respect to specific examplesincluding presently preferred modes of carrying out the invention, thoseskilled in the art will appreciate that there are numerous variationsand permutations of the above described systems and techniques that fallwithin the spirit and scope of the invention as set forth in theappended claims.

EXAMPLES Example 1

The following reagents were used in screening and characterizing mutantstrains:

-   -   5-bromo-4-chloro-3-indolyl sulfate (X—SO₄); CAS No. 6578-07-0        Source: Sigma.    -   5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-GlcA); CAS No.        114162-64-0, Source: Sigma or RPI Corp.

Example 2

The genes encoding arylsulfatase and β-glucuronidase were identified bymaking a library of random transposon mutants in a nonrmucoid strain ofS. elodea, Gps2, using the commercially available EZ::TN™ <R6Kgamma-ori/KAN-2> insertion kit from Epicentre (Madison, Wis.).Kanamycin resistant mutant strains were screened for lack of (orsignificantly reduced) blue color on selective media with specificchromogenic substrates. See Example 1. Mutants blocking arylsulfataseproduction or activity were identified using the chromogen5-bromo-4-chloro-3-indolyl sulfate (X—SO4) on agar with a defined mediumwith chloride salts. Mutants of β-glucuronidase were selected on adefined medium with the chromogen5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-GlcA).

The transposon and adjacent genomic DNA were subsequently excised fromthe chromosome using restriction enzymes. The restriction enzymefragments were circularized with ligase and transformed into Escherichiacoli where the transposon-containing DNA can replicate due to presenceof a replicon in the transposon. Plasmid DNA was purified and sequenced.The plasmid with the gene for arylsulfatase was designated R6K-AS#14E. Aportion of this plasmid has been sequenced (SEQ ID NO: 1). The plasmidwith the gene for beta-glucuronidase was designated R6K-BG#6S. A portionof this plasmid has been sequenced (SEQ ID NO: 4.) These plasmids inEscherichia coli strain EC100D pir+ are being deposited at the AmericanType Culture Collection, Manassas, Va., on Jun. 21, 2004.

In the bacterium that had an inactivated arylsulfatase, the transposonhad actually inserted in a gene for a hypothetical protein that wasadjacent to the gene for arylsulfatase. In the bacterium that had aninactivated β-glucuronidase, the transposon insertion was located in theamino portion of the gene for β-glucuronidase. DNA sequencing of thegenes showed that they were homologous to known genes from other speciesin the database of the National Center for Biotechnology Information(NCBI).

The genes for arylsulfatase and β-glucuronidase and adjacent genomic DNAwere sequenced. Deletions of the genes were constructed on a plasmid andthen transferred into S. elodea strains S-60 wtc and PDG-1. See WO01/64897. The deletions were inserted in the genome by homologousrecombination. DNA sequences flanking the target gene were amplified byPCR and cloned into the plasmid vector pLO2. (Lenz, O., E. Schwartz, J.Demedde, M. Eitinger and B. Friedrich. 1994, “The Alcaligenes eutrophusH16 hoxX gene participates in hydrogenase regulation,” J. Bacteriol.176:4385-4393.) This construct was transferred into the S. elodea strainby conjugation. The resulting kanamycin resistant strains were thengrown for 30-40 generations in the absence of antibiotic. Isolates thathad lost the plasmid were detected by selection for sucrose tolerancedue to loss of the sacB gene on pLO2, and confirmed by kanamycinsensitivity. Isolates that had lost the plasmid after the non-selectivegrowth were of two types, deletion or wild-type. The desired deletionstrains were identified by lack of blue color on appropriate indicatoragar (see example 1) and by diagnostic PCR. The scheme for constructionis shown in FIG. 1 below.

To construct a precise deletion of the gene (atsA) for arylsulfatase itwas necessary to determine the most likely start codon for the atsA geneand the start and stop codons of the adjacent genes. The locations ofthe ends of the genes were determined based on DNA sequences, homologiesto other genes in GenBank and third base GC preference using theFramePlot-3 program. Ishikawa and Hotta. “FramePlot: a newimplementation of the Frame analysis for predicting protein-codingregions in bacterial DNA with a high G+C content.” FEMS MicrobiologyLetters 174:251-253 (1999). This analysis indicated that thearylsulfatase gene is translationally coupled to the gene for theconserved hypothetical protein, i.e., the stop codon of thearylsulfatase gene overlaps with the start codon of the gene for theconserved hypothetical protein. The arylsulfatase deletion wasconstructed to leave the alkylsulfatase gene and the gene for theunknown protein intact, since it is not known whether these proteins arerequired for optimal cell growth and gellan production.

PCR primers AS5 (CCGAGCTCAACGCCTTCGACTATGTCCA; SEQ ID NO: 11) and AS6(CCTCTAGACTGGGGATTGTCCGGAAAAG; SEQ ID NO: 12) were used to amplify a 512bp fragment just upstream of the start codon of atsA as a SacI-XbaIfragment (total 528 bp). Primers AS3 (CGTCTAGATCCACCCCGGCGACCTTCCC; SEQID NO: 13) and AS4 (TATAGCATGCGGCGACCACGGGCTCCTCCTCA; SEQ ID NO: 14)were used to amplify a 479 bp fragment including the end of the atsAgene and the start of the conserved hypothetical protein as an XbaI-SphIfragment (total 497 bp). Thus, the stop codon of atsA was retained butthe start codon was deleted, so no portion of the arylsulfatase proteinshould be synthesized. Restriction sites for cloning were added to theends of the primers. The PCR fragments were ligated sequentially intothe polylinker of plasmid vector pLO2, to form the deletion of the atsAgene. The upstream SacI-XbaI fragment was cloned into SacI-XbaI cutpLO2. Subsequently the downstream XbaI-SphI fragment was cloned (FIG.2). This plasmid with deletion of the atsA gene was transferred byconjugation into S. elodea strains S60 wtc and PDG-1 to allowrecombination of the plasmid into the chromosome. Kanamycin resistantisolates were purified, then grown in the absence of antibiotic andplated on medium with sucrose and X—SO₄ to select isolates with sucrosetolerance due to loss of the plasmid-encoded sacB gene. Sucroseresistance, kanamycin sensitive, yellow colonies were selected. The atsAderivatives of S60 wtc and PDG-1 were designated GAS-1 and PAS-1respectively.

A deletion of the gene (gusA) for β-glucuronidase was constructed onplasmid pLO2 and transferred into S60 wtc, GAS-1. The most likely startcodon for the gusA gene was determined by homology to other proteins andthe presence of ribosome binding sites. A region of secondary structureis upstream of the start codon. The deletion of the gusA gene wasconstructed to maintain secondary structures upstream and downstream ofgusA. Primers Bgluc3 (AACTGCAGACACGTGGCTTGTGCCGAAC; SEQ ID NO: 7) andBgluc4 (GGCTCTAGACTTCTCCCTGTTCCTCCGGGAAA; SEQ ID NO: 8) were used toamplify a 560 bp fragment upstream of the gusA gene as a PstI-XbaIfragment (total 577 bp). Primers Bgluc1 (TTTCTAGATGACTGTCCAGGCCCCTCTC;SEQ ID NO: 9) and Bgluc2 (TCGAGCTCCAATGTCCTCGTAGCTGTTC; SEQ ID NO: 10)were used to amplify a 489 bp fragment downstream of gusA as anXbaI-SacI fragment (total 505 bp). The PstI-XbaI fragment was clonedinto PstI-XbaI cut pLO2. Subsequently the downstream XbaI-SacI fragmentwas cloned. This plasmid with deletion of the gusA gene was transferredby conjugation into S. elodea strains S60 wtc, GAS-1 and PAS-1 to allowrecombination. Kanamycin resistant isolates were purified, grown in theabsence of antibiotic and then plated on media with sucrose and X-GlcAto select isolates with sucrose tolerance due to loss of theplasmid-encoded sacB gene. A mixture of blue-green (wild-type) and lightgreen (mutant) colonies was obtained. Sucrose resistant, light greenisolates were confirmed for plasmid loss by kanamycin sensitivity. ThegusA deletion derivatives of S60 wtc, GAS-1 and PAS-1 were designatedGBG, GBAD and PBAD respectively.

The deletion of the gene (atsA) for arylsulfatase in strains S60 wtc andPDG-1 has been completed. The gene for β-glucuronidase has beenidentified and sequenced. The adjacent DNA was sequenced. A deletion ofthe gene for β-glucuronidase has also been constructed in each of S60,PDG-1, and GAS-1.

Samples of gellan made from strains GAS-1 (with a deletion of the genefor arylsulfatase) and GBAD-1 (with deletions of genes for botharylsulfatase and β-glucuronidase) were evaluated for p-cresolproduction at monthly intervals in an ultra-high temperature dairyapplication test. Gellan samples from the wild-type strain produced 3 to152 (average 65) ppb p-cresol after one month and 4 to 212 (average 96)ppb p-cresol after two months. Samples of gellan from GAS-1 producedabout 1 to 3 ppb p-cresol after one to five months. Samples of gellanfrom GBAD-1 produced less than 1 ppb (limit of detection) when testedfor up to three months.

1. A composition comprising genetically purified gellan gumsubstantially free of catalytically active arylsulfatase protein andsubstantially free of denaturing agent and substantially free ofdenatured arylsulfatase protein.
 2. A composition comprising geneticallypurified gellan gum substantially free of catalytically activeβ-glucuronidase protein and substantially free of denaturing agent andsubstantially free of denatured β-glucuronidase protein.
 3. Acomposition comprising genetically purified gellan gum substantiallyfree of catalytically active arylsulfatase and catalytically activeβ-glucuronidase proteins and substantially free of denaturing agent andsubstantially free of denatured arylsulfatase and β-glucuronidaseprotein.
 4. The composition of claim 1 which is a culture broth ofSphingomonas elodea.
 5. The composition of claim 1 which is aprecipitated culture broth of Sphingomonas elodea.
 6. The composition ofclaim 1 which is a sterilized milk product.